Based upon the observation that aquaporin-4 (AQP4)-specific antibodies are IgG1, a T cell-dependent isotype, it was hypothesized that aquaporin-4 (AQP4)-specific T cells have a key role in neuromyelitis optica (NMO) pathogenesis. AQP4-specific T cells have been identified in NMO patients and, in comparison to healthy controls (HC), AQP4-reactive T cells are expanded and exhibit Th17 polarization, findings that further support the role of Th17 cells in NMO. Unfortunately, it is not feasible to evaluate how AQP4-reactive T cells participate directly in CNS inflammation in NMO patients. Thus, it is important to develop models to evaluate the potential role of AQP4-specific T cells in NMO. Initial attempts in generating an in vivo AQP4-based NMO model in wild-type (WT) mice and rats have not met with success. Recently, it was observed that pathogenic AQP4-specific T cells exist in AQP4-deficient (AQP4-/-) mice. Those T cells recognize two novel determinants. In comparison to other AQP4 determinants identified in WT mice, the novel determinants induce robust proliferation in AQP4-/- mice, but only a weak response in WT mice. Hyper-reactivity is AQP4-specific, but not epitope-specific as we discovered a second AQP4 epitope induced vigorous proliferation in AQP4- /-, but not WT, mice. The T cell receptor (TCR) repertoires used for recognition of these determinants in AQP4-/- and WT mice are distinct. T cells reactive to these determinants isolated from AQP4-/- donor mice induced clinical and histologic CNS autoimmune disease in 100% of recipient WT mice tested. Collectively, these findings represent the first successful induction of clinical AQP4-targeted CNS autoimmunity. Our findings suggest responses to those epitopes in AQP4-/- mice reflect a loss of central T cell tolerance. Findings from studying mice deficient in T cells only or B cells only indicate that peripheral T cell regulation also alters expression of pathogenic AQP4-specific T cells. We propose to test our hypothesis that there is a defect in thymic negative selection of AQP4-specific T cells in mice, a possibility that may be relevant to NMO pathogenesis. We will examine how peripheral T cell regulation may influence pathogenic AQP4-specific immune responses. In Specific Aim 1, by using unique approaches and novel mice, we will characterize the phenotype of pathogenic AQP4-specific T cells and generate AQP4-specific TCR transgenic mice. In Specific Aim 2, we will characterize the repertoire of AQP4-specific T cells in AQP4-/- and WT mice that express HLA- DR17 (DRB1*0301), the MHC II allele most highly associated with NMO. Separately, we will examine AQP4-specific T cell responses in NMO patients to determine if DR17-restricted AQP4-specific T cell epitopes identified those mice correspond to AQP4 T cell epitopes in NMO patients. In preliminary data, using these mice we discovered a novel HLA- DR17-restricted AQP4 determinant, and observed that it is recognized by T cells from HLA-DR17+ NMO patients, findings that support feasibility of this aim. Findings obtained in this program should elucidate mechanisms controlling development of pathogenic AQP4-specific T cells in NMO, provide a foundation advancing development of in vivo NMO models, and may provide insight for development of selective NMO immunotherapy.